Methods for detecting asymmetric dimethylarginine in a biological sample

ABSTRACT

The present invention provides methods of detecting asymmetric dimethylarginine (ADMA) in a sample, particularly a sample that may contain symmetrical dimethylarginine (SDMA) and/or arginine. The methods generally involve modifying any SDMA and arginine in the sample such that SDMA and arginine are readily distinguishable from ADMA; and detecting ADMA. The invention further provides antibodies specific for ADMA; antibodies specific for modified SDMA; and antibodies specific for modified arginine. The invention further provides kits for practicing the subject methods.

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/426,677 filed Nov. 15, 2002, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The U.S. government may have certain rights in this invention,pursuant to grant no. R01 HL-63685 awarded by the National Institutes ofHealth, the National Heart, Lung and Blood Institute.

FIELD OF THE INVENTION

[0003] The present invention is in the field of assay methods, and inparticular assay methods for asymmetric dimethylarginine.

BACKGROUND OF THE INVENTION

[0004] Elevated asymmetric dimethylarginine (ADMA) levels have beenobserved in various conditions, including hypertension, dyslipidemia,hyperglycemia, hyperhomocysteinemia, and renal failure, and are believedto be one cause of endothelial dysfunction in these conditions. Elevatedplasma ADMA concentrations are also associated with an increased risk ofcardiovascular disease. As an endogenous inhibitor of nitric oxidesynthase, ADMA reduces nitric oxide (NO) production. NO plays a vitalpart in the vascular homeostasis. Aside from being the most potentvasodilator, NO inhibits platelet aggregation, smooth muscleproliferation, and adhesion molecule expression, which all play a partin atherogenesis. Throughout the last few years, basic scientificinvestigation has revealed the mechanism whereby ADMA becomes elevatedin patients with hypertension, hyperhomocysteinemia, hyperglycemia,hypercholesterolemia, and tobacco exposure.

[0005] Nevertheless, the field of ADMA is progressing slowly, mostlybecause of the laborious procedures required to quantify the molecule. Aconclusive demonstration of ADMA's clinical relevance requires clinicalstudies with a great patient population. High pressure liquidchromatography (HPLC) is the most commonly used method to quantify ADMA.However, the use of HPLC to quantitate ADMA suffers from severaldrawbacks. The most critical among them are efficiency and sensitivity.The labor-intensive extraction and derivatization steps necessary forHPLC detection not only makes the procedure more vulnerable to humanerrors, but also makes it unfitting for studies with a large samplesize. Moreover, because the detection limit for UV detectors seldom goesbelow the sub-micromolar level, intracellular ADMA level in diseasestates remains largely unexplored, despite the fact that ADMA isgenerated intracellularly.

[0006] There is a need in the art for methods of detecting andquantitating ADMA that are simple, efficient, and readily adapted tohigh-throughput analysis. The present invention addresses this need.

[0007] Literature

[0008] Takahashi (1968) J. Biol. Chem. 243:6171-6179; Stühlinger et al.(2002) J. Am. Med. Assoc. 287:1420-1426; U.S. Pat. No. 6,358,699;Teerlink et al. (2002) Anal. Biochem. 303:131-137; Dobashi et al. (2002)Analyst 127:54-59; Vishwanathan et al. (2000) J. Chromatogr. B. Biomed.Sci. Appl. 748:157-166; Pi et al. (2000) J. Chromatogr. B. Biomed. Sci.Appl. 742:199-203; Chen et al. (1997) J. Chromatogr. B. Biomed. Sci.Appl. 692:467-471; Pettersson et al. (1997) J. Chromatogr. B. Biomed.Sci. Appl. 692:257-262.

SUMMARY OF THE INVENTION

[0009] The present invention provides methods of detecting asymmetricdimethylarginine (ADMA) in a sample, particularly a sample that maycontain symmetrical dimethylarginine (SDMA) and/or arginine. The methodsgenerally involve modifying any SDMA and arginine in the sample suchthat SDMA and arginine are readily distinguishable from ADMA; anddetecting ADMA. The invention further provides antibodies specific forADMA; antibodies specific for modified SDMA; and antibodies specific formodified arginine. The invention further provides kits for practicingthe subject methods.

FEATURES OF THE INVENTION

[0010] The present invention features a method of detecting asymmetricdimethylarginine (ADMA) in a sample comprising ADMA, symmetricdimethylarginine (SDMA), and arginine. The method generally involves: a)contacting a sample with an α-dicarbonyl compound, wherein said sampleis suspected of containing ADMA and at least one of SDMA and arginine,where the contacting step results in modification of the guanidinonitrogens of SDMA and the guanidino nitrogens of arginine, to producemodified SDMA and modified arginine; and b) detecting ADMA in thesample. In some embodiments, the α-dicarbonyl compound is phenylglyoxal.

[0011] In some embodiments, the method further involves the step ofmodifying the α-amino group of SDMA, ADMA, and arginine before the stepof modifying the guanidino nitrogens of SDMA and the guanidino nitrogensof arginine. In some of these embodiments, the α-amino group is modifiedwith a dye that provides a detectable signal.

[0012] In some embodiments, the detection step involves contacting thesample with an antibody that binds specifically to dimethylarginines,wherein the antibody does not bind to the modified SDMA. In otherembodiments, the detection step involves contacting the sample with anantibody that binds specifically to ADMA. In some of these embodiments,the antibody is detectably labeled.

[0013] In some embodiments, detection of ADMA is by high performanceliquid chromatography. In other embodiments, detection of ADMA is bycapillary electrophoresis.

[0014] The present invention further features an antibody that bindsspecifically to asymmetric dimethylarginine. In some embodiments, theantibody is detectably labeled.

[0015] The present invention further features an antibody that bindsspecifically to modified symmetric dimethylarginine (SDMA), wherein theguanidino nitrogens of the SDMA are modified by reaction with anα-dicarbonyl compound.

[0016] The present invention further features a kit for detectingasymmetric dimethylarginine (ADMA) in a sample. In some embodiments, thekit includes an α-dicarbonyl agent that modifies the guanidino nitrogensof SDMA and the guanidino nitrogens of arginine; and an antibody thatbinds to ADMA. In other embodiments, the kit further includes anantibody that binds α-dicarbonyl-modified SDMA, and an antibody thatbinds α-dicarbonyl-modified L-arginine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 depicts the structure of phenylglyoxal.

[0018]FIG. 2 depicts the reaction of phenylglyoxal with an arginineresidue.

[0019]FIG. 3 depicts fluoro-nitro-benzoxadiazole labeled ADMA.

[0020]FIG. 4 depicts the reaction of SDMA with phenylglyoxal.

DEFINITIONS

[0021] Assay methods of the invention may be qualitative orquantitative. Thus, as used herein, the term “detection” refers to bothqualitative and quantitative determinations, and therefore includes“measuring” and “determining a level of.”

[0022] A “biological sample” encompasses a variety of sample typesobtained from an individual and can be used in a diagnostic ormonitoring assay. The definition encompasses blood, blood-derivedsamples, and other liquid samples of biological origin, solid tissuesamples such as a biopsy specimen or tissue cultures or cells derivedtherefrom and the progeny thereof. The definition also includes samplesthat have been manipulated in any way after their procurement, such asby treatment with reagents, solubilization, or enrichment for certaincomponents. The term “biological sample” encompasses a clinical sample,and also includes cells in culture, cell supernatants, cell lysates,serum, plasma, cerebrospinal fluid, urine, saliva, biological fluid, andtissue samples.

[0023] The term “binds specifically,” in the context of antibodybinding, refers to high avidity and/or high affinity binding of anantibody to a specific molecule, e.g., asymmetric dimethylarginine(ADMA), modified symmetric dimethylarginine (SDMA), or modifiedarginine. For example, binding of an ADMA-specific antibody to ADMA isstronger than binding of the same antibody to arginine, SDMA, ormodified SDMA, so that by adjusting binding conditions, the antibodybinds almost exclusively to ADMA and not to arginine, SDMA, modifiedarginine, or modified SDMA. Likewise, binding of an antibody specific tomodified SDMA is stronger than binding of the same antibody to arginine,ADMA, modified ADMA, or SDMA, so that by adjusting binding conditions,the antibody binds almost exclusively to SDMA and not to arginine, ADMA,modified ADMA, or SDMA.

[0024] Antibodies which bind specifically to ADMA, to modified SDMA, orto modified arginine may be capable of binding other molecules at aweak, yet detectable, level (e.g., 10% or less of the binding shown toADMA, modified SDMA, or modified arginine). Such weak binding, orbackground binding, is readily discernible from the specific antibodybinding to ADMA, modified SDMA, or modified arginine, e.g. by use ofappropriate controls. In general, antibodies of the invention which bindto ADMA, modified SDMA, or modified arginine, with a binding affinity of10⁻⁷ mole/l or more, e.g., 10⁻⁸ mole/liters or more (e.g., 10⁻⁹ M,10⁻¹⁰, 10⁻¹¹, etc.) are said to bind specifically to ADMA, modifiedSDMA, or modified arginine, respectively. In general, an antibody with abinding affinity of 10⁻⁶ mole/liters or less is not useful in that itwill not bind an antigen at a detectable level using conventionalmethodology currently used.

[0025] Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

[0026] Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

[0027] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

[0028] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“an α-dicarbonyl compound” includes a plurality of such compounds andreference to “the modified SDMA” includes reference to one or moremodified SDMA molecules and equivalents thereof known to those skilledin the art, and so forth.

[0029] The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides methods of detecting asymmetricdimethylarginine (ADMA) in a sample, particularly in a sample that maycontain SDMA and/or arginine. The methods generally involve modifyingsymmetrical dimethylarginine (SDMA) and arginine such that SDMA andarginine are readily distinguishable from ADMA; and detecting ADMA. Theinvention further provides antibodies specific for ADMA, antibodiesspecific for modified SDMA, and antibodies specific for modifiedarginine. The invention further provides kits for practicing the subjectmethods.

Methods of Detecting ADMA in a Biological Sample

[0031] The present invention provides methods of detecting ADMA in abiological sample, particularly in a sample that may contain SDMA and/orarginine. The biological sample may comprise ADMA, SDMA, and arginine.The methods involve modifying SDMA and arginine in such a way that themodified SDMA and modified arginine are readily distinguishable fromADMA. The methods involve contacting a biological sample with anα-dicarbonyl compound, generating modified SDMA and modified arginine;and detecting ADMA in the sample.

[0032] The structures of arginine, ADMA, and SDMA are shown below. Theinstant methods involve modifying the guanidino nitrogen groups of SDMAand of arginine with an α-dicarbonyl compound. SDMA and arginine aremodified with the α-dicarbonyl compound, while ADMA is not.

[0033] Acting as nucleophiles, the guanidino nitrogens of arginineattack the carbonyl carbons of the α-dicarbonyl compound, generating amodified arginine that contains two modified nitrogen groups perguanidino group. Since the guanidino nitrogens on SDMA each take up onemethyl group, they both still possess a hydrogen that is free toparticipate in a nucleophilic reaction. Thus, as with arginine, theguanidino nitrogens of SDMA are also modified by the α-dicarbonylcompound to form a stable product. Without wishing to be bound bytheory, it is believed that participation of both guanidino nitrogenswith the α-dicarbonyl compound is crucial for modification of thecompound because the resultant 5-membered ring structure stabilizes theintermediate product. Because ADMA has both methyl groups occupying thesame guanidino nitrogen, the guanidino nitrogen is not available forreacting with the α-dicarbonyl compound, and ADMA does not react with anα-dicarbonyl compound to form a stable product.

[0034] Modifying SDMA and Arginine

[0035] Any of a variety of α-dicarbonyl compounds that are known in theart can be used in the instant methods to modify guanidino nitrogens ofSDMA and arginine. The structure of a generic α-dicarbonyl compound isshown below.

[0036] Suitable α-dicarbonyl compounds include, but are not limited to,dialdehydes, ketoaldehydes, and diketones. Non-limiting examples ofα-dicarbonyl compounds are biacetyl, pyruvic acid, glyoxal,methyglyoxal, deoxyosones, 3-deoxyosones, malondialdehyde,2-oxopropanal, phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione.

[0037] In many embodiments, R is a bulky group, including, but notlimited to, a cyclopentyl group, a substituted cyclopentyl group; asix-membered ring, such as phenyl, a substituted phenyl (e.g.,p-hydroxyphenylglyoxal, nitrophenylglyoxal, etc.), and the like. Inembodiments of particular interest, the α-dicarbonyl compound isphenylglyoxal. The structure of phenylglyoxal is shown below.

[0038] As one non-limiting example, where the α-dicarbonyl compound isphenylglyoxal, the reaction with arginine proceeds as follows:

[0039] The reaction with SDMA proceeds in a similar way.

[0040] In addition to reacting with the guanidino amine of arginine,phenylglyoxal has been reported to react with the α-amino group of thepeptides to give α-keto acyl peptides. Takahashi (1968) J. Biol. Chem.243:6171-6179. In the context of free amino acid, this observationindicates that phenylglyoxal will react with all α-amino groups of allamino acid. In some embodiments, the α-amino group is protected with aconventional labeling dye such as fluoro-nitro-benzoxadiazole (NBD-F),as described further below. Protection of the α-amino group ensures thatphenylglyoxal only reacts with the guanidino nitrogen. Many labeling dyemolecules are conjugated to the amino acid or proteins by reacting withthe α-amino group.

[0041] An α-dicarbonyl compound is contacted with a biological sample.Generally, the α-dicarbonyl compound is prepared in water, and the pH ofthe solution is adjusted to 9.0 with 1M NaOH. The α-dicarbonyl compoundis generally in a 10× stock solution in a concentration of from about 1mM to about 500 mM, e.g., from about 1 mM to about 10 mM, from about 10mM to about 50 mM, from about 50 mM to about 100 mM, from about 100 mMto about 200 mM, from about 200 mM to about 300 mM, from about 300 mM toabout 400 mM, or from about 400 mM to about 500 mM. In some embodiments,the α-dicarbonyl compound is in a 10× stock solution in a concentrationof from about 50 mM to about 100 mM. A solution containing theα-dicarbonyl compound is added to the biological sample in such a waythat the stock solution is diluted 10-fold.

[0042] The biological sample is contacted with the α-dicarbonylcompound, and the reaction is allowed to proceed for a period of time offrom about 15 seconds to 2 hours, e.g., from about 15 seconds to about60 seconds, from about 1 minute to about 15 minutes, from about 15minutes to about 30 minutes, from about 30 minutes to about 60 minutes,or from about 1 hour to about 2 hours. In a particular embodiment, thereaction is allowed to proceed for 1 hour to about 2 hours in the darkat room temperature (e.g., at about 22° C.).

[0043] The reaction of components in the sample with the α-dicarbonylcompound results in modification of the guanidino nitrogen groups ofsubstantially all molecules of SDMA and arginine in the sample. Thus, atleast 80%, at least 90%, at least 95%, at least 98%, or at least 99% ofthe SDMA and arginine molecules in the sample are modified.

[0044] A variety of other reagents may be included in the assay. Theseinclude reagents such as buffers, salts, neutral proteins, e.g. albumin,detergents, etc. Reagents that improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may also be used.

[0045] The instant assay methods may be designed a number of differentways. For example, the assay components of the method may be combined atsubstantially the same time or at different times. Incubations areperformed at any suitable temperature, typically between 4° and 40° C.Incubation periods are selected for optimum activity, but may also beoptimized to facilitate rapid high-throughput screening. In general, allcomponents are in solution.

[0046] An example of a biological sample is serum. However, anybiological sample can be used. As one non-limiting example, serum orplasma is obtained from a blood sample of a patient, and from about 0.05mL to about 2.0 mL of serum or plasma is reacted with an α-dicarbonylcompound such that the final concentration of the α-dicarbonyl compoundin the sample is in a range of from about 30 mM to about 70 mM. Thereaction is allowed to proceed for a suitable time, after which ADMA isdetected.

[0047] Protecting the α-Amino Group

[0048] The α-dicarbonyl compound may react with the α-amino group ofarginine, SDMA, and ADMA. In such cases, an optional step ofderivatizing the α-amino group of arginine, SDMA, and ADMA is performedbefore the modification of the guanidino nitrogen groups of arginine andSDMA. Many methods are known in the art for modifying (protecting) theα-amino group.

[0049] Protecting groups for α-amino groups are well known in the artinclude, but are not limited to, methyl, formyl, ethyl, acetyl, t-butyl,anisyl, benzyl, trifluoroacetyl, N-hydroxysuccinimide,t-Butyloxycarbonyl, benzoyl, 4-methylbenzyl, thioanizyl, thiocresyl,benzyloxymethyl, 4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl,2-nitrophenylsulphenyl, 4-toluenesulfphonyl, pentafluorophenyl,diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl,2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl,2,2,5,7,8-pentamethyl-chroman-6-sulphonyl, 2-(4-nitrophenylsulfonyl)ethoxy carbonyl, 9-(2-sulfo)fluorenylmethyl carbamate,9-(2,7-dibromo)fluorenylmethyl carbamate,17-tetrabenzol[a,c,g,i]fluorenyl methyl carbamate,1,1-dioxobenzol[b]thiophen-2-ylmethyl carbamate, and the like.

[0050] Also suitable for use are groups that both protect the α-aminogroup and provide for detection, e.g., the protecting group is adetectable label. Suitable fluorophores for this invention includefluorescein and its analogs, rhodamine and its analogs, cyanine andrelated polymethines and their analogs, and the like. Specificfluorophores which are suitable for use with the present invention areFluorescein isothiocyanate (FITC); 4-fluoro-7-nitrobenzofurazan (NBD-F);Texas Red™ (Molecular Probes, Inc.; Eugene, Oreg.); tetramethylrhodamine isothiocyanate (TRITC); and Cyanine dyes, e.g., Cy3, Cy5,Cy5.5 Cy7, Cy7.5, Cy8 and Cy9 (Biological Detection Systems, Pittsburgh,Pa.); phenyl-thiohydantoin (PTH), and phenylisothiocyanate (PITC). Asone non-limiting example (see above), the α-amino group is reacted with4-fluoro-7-nitro-benzoxadiazole (NBD-F).

[0051] Detecting ADMA

[0052] ADMA is detected using any of a variety of methods. Such methodsinclude, but are not limited to, conversion of ADMA to citrulline,followed by spectrophotometric determination of citrulline;determination of ADMA with an antibody that binds dimethylarginines;determination of ADMA with an antibody specific for ADMA, using animmunological assay; high performance liquid chromatography; capillaryelectrophoresis; and the like. Detection of ADMA can be qualitative orquantitative. These methods are also useful for detecting derivatizedSDMA and L-arginine, which, after reacting as described above, are moreeasily distinguished from ADMA. Various methods are outlined below.

[0053] HPLC Methods

[0054] A variety of methods for detecting ADMA using HPLC are known inthe art, any of which can be used in conjunction with an instant assaymethod. See, e.g., Teerlink et al. (2002) Anal. Biochem. 303:131-137;Dobashi et al. (2002) Analyst 127:54-59; Pi et al. (2000) J. Chromatogr.B. Biomed. Sci. Appl. 742:199-203; Chen et al. (1997) J. Chromatogr. B.Biomed. Sci. Appl. 692:467-471; Anderstam et al. (1997) J. Am. Soc.Nephrol. 8:1487-1442; and Pettersson et al. (1997) J. Chromatogr. B.Biomed. Sci. Appl. 692:257-262.

[0055] Where HPLC is used to detect ADMA in a sample, the α-amino groupof ADMA is modified (derivatized) with a detectable group, e.g., afluorescent group. After the α-amino group is derivatized, the sample istreated with an α-dicarbonyl compound as described above. This willcause SDMA and arginine, but not ADMA, to be modified, so as to enhancetheir separation from ADMA when the sample is applied to an HPLC column.Separation of analytes is performed on the HPLC using a solvent. Theanalytes, including ADMA, are eluted, and the amount of ADMA isdetermined by measuring the amount of the detectable label in eachanalyte peak. The amount of ADMA can be determined by determining thepeak area.

[0056] Solid phase extraction (SPE) of the biological sample isfrequently carried out to clean up the sample prior to applying thesample to the HPLC column. A variety of SPE columns are available andcan be used in conjunction with the instant methods. Suitable SPEcolumns include an Oasis MCX SPE column (Waters); a Bond Elute SCX 50 mgcolumn (Varian Inc., Palo Alto, Calif.); and the like. These columnsretain positively-charged compounds, which are then collected by elutingthe column with a weak base such as trimethylamine.

[0057] The following is one non-limiting example of a method ofdetecting ADMA using HPLC. In this method, the biological sample iscleaned up on an SPE column; the primary amine containing compounds (eg.ADMA, SDMA, arginine) are derivatized with an ortho-phthaldialdehydereagent containing 3-mercaptopropionic acid; and, after thisderivatization, the sample is treated with an α-dicarbonyl compound andthen the derivatized sample is applied to a reversed-phase HPLC, and thepeak corresponding to ADMA is detected with fluorescence detection. Thepeak corresponding to ADMA is clearly distinguished from peakscorresponding to L-arginine or SDMA.

[0058] In this example, the biological sample is first cleaned up on aSPE column. 0.2 ml of biological sample (e.g., plasma, serum, urine,cerebrospinal fluid, and the like) is mixed with 0.1 ml of an internalstandard and 0.7 ml phosphate buffered saline. The sample is applied toan Oasis MCX SPE column (Waters). After application of the sample, thecolumn is washed consecutively with 1.0 ml of 100 mM HCl and 1.0 mlmethanol. Analytes are eluted from the column in 3.0-ml tubes with 1.0ml concentrated ammonia (or triethylamine)/water/methanol (10/40/50).The solvent is removed from the analytes by evaporation with nitrogen ata temperature of 60-80° C. The residue is dissolved in 0.1 ml water toform the analyte sample.

[0059] In this example, analytes eluted from the SPE column arederivatized with ortho-phthaldialdehyde (OPA). To the 0.1 ml analytesample is added 0.1 ml OPA working solution. OPA stock solution isprepared by dissolving 10 mg OPA in 0.2 ml methanol, followed byaddition of 1.8 ml of a 200 mM potassium borate buffer (pH 9.5) and 10μl 3-mercaptopropionic acid. The OPA working solution is prepared byfive-fold dilution of the stock solution with borate buffer. The finalconcentrations of OPA and 3-mercaptopropionic acid in the working OPAsolution are 7.5 mM and 11.5 mM, respectively. After the OPA workingsolution is added to the analyte sample, the samples are derivatizedwith an α-dicarbonyl compound, and are applied to an HPLC column.

[0060] In this example, HPLC is performed on a Symmetry C18 column(3.9×150 mm; 5 μm particle size; 100 Å pore size) with a 10×3 mm guardcolumn packed with the same stationary phase. Mobile phase A consists of50 mM potassium phosphate buffer (pH 6.5), containing 8.7% acetonitrile,and mobile phase B is acetonitrile/water (50/50, v/v). Separation isperformed under isocratic conditions with 100% mobile phase A at a flowrate of 1.1 muminute and a column temperature of 30° C. After elution ofthe last analyte, closely related compounds are eluted with a strongsolvent flush (50% B from 20 to 22 minutes). Fluorescence is measured atexcitation and emission wavelengths of 340 an 455 nm, respectively.Peaks are quantified on the basis of peak area.

[0061] Those skilled in the art will recognize that many modificationsto the above example of an HPLC method are possible.

[0062] Capillary Electrophoresis

[0063] Where CE is used to detect ADMA content of a sample, α-aminogroups of all of the amino acids (including that of ADMA) are modifiedwith a labeling group (e.g., NBD-F). Subsequently, SDMA and L-arginineare derivatized with an α-dicarbonyl compound.

[0064] Methods of using capillary electrophoresis to detect ADMA areknown in the art, and any known method can be used in conjunction withthe instant methods. See, e.g., Vallance et al. (1992) Lancet339:572-575; and Causse et al. (2000) J. Chromatogr. B. Biomed. Sci.Appl. 741:77-83.

[0065] Immunoassays

[0066] A variety of immunoassays can be designed that make use of theability to distinguish modified SDMA and modified arginine from ADMA. Incarrying out an immunoassay, one or more of the following antibodieswill be used: (1) antibodies that bind dimethylarginines, i.e., thatspecifically bind both SDMA and ADMA and that do not discriminatebetween SDMA and ADMA; (2) antibodies that specifically bind ADMA; (3)antibodies that specifically bind modified SDMA; (4) antibodies thatbind both modified SDMA and modified L-arginine; and (5) antibodies thatspecifically bind modified arginine.

[0067] Modification of arginine and SDMA as described above modifiesthese molecules such that they no longer react with antibodies that bindboth SDMA and ADMA (i.e., antibodies specific for dimethylarginines).Thus, in some embodiments, determination of ADMA following modificationof arginine and SDMA is carried out with conventional immunologicalassays, using antibodies specific for dimethylarginines. Becausesubstantially all of the SDMA in the sample is modified by theα-dicarbonyl compound during the modification reaction, and because themodified SDMA is not recognized by antibodies specific fordimethylarginines, such antibodies will only detect ADMA in the sample.

[0068] Detection of ADMA can also be carried out using an antibodyspecific for ADMA (e.g., antibody that binds ADMA, but that does notsubstantially bind to SDMA, arginine, or modified SDMA), as described inmore detail below.

[0069] Immunoassays can also be carried out using antibodies specificfor modified SDMA and antibodies specific for modified arginine, asdescribed in more detail below.

[0070] Detection with a specific antibody is carried out usingwell-known methods. In general, the antibody is detectably labeled,either directly or indirectly. Direct labels include radioisotopes(e.g., ¹²⁵I; ³⁵S, and the like); enzymes whose products are detectable(e.g., luciferase, β-galactosidase, horse radish peroxidase, and thelike); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine,phycoerythrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu,or others of the lanthanide series, attached to the antibody throughmetal chelating groups such as EDTA; chemiluminescent compounds, e.g.,luminol, isoluminol, acridinium salts, and the like; bioluminescentcompounds, e.g., luciferin; fluorescent proteins; and the like.Fluorescent proteins include, but are not limited to, a greenfluorescent protein (GFP), including, but not limited to, a “humanized”version of a GFP, e.g., wherein codons of the naturally-occurringnucleotide sequence are changed to more closely match human codon bias;a GFP derived from Aequoria victoria or a derivative thereof, e.g., a“humanized” derivative such as Enhanced GFP, which are availablecommercially, e.g., from Clontech, Inc.; a GFP from another species suchas Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, asdescribed in, e.g., WO 99/49019 and Peelle et al. (2001) J. ProteinChem. 20:507-519; “humanized” recombinant GFP (hrGFP) (Stratagene); anyof a variety of fluorescent and colored proteins from Anthozoan species,as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973;and the like.

[0071] Indirect labels include second antibodies that bind to antibodiesspecific for ADMA or dimethylarginines, wherein the second antibody islabeled as described above. Indirect labels also include members ofspecific binding pairs, e.g., biotin-avidin, and the like, as are wellknown in the art. For example, the primary antibody may be conjugated tobiotin, with horseradish peroxidase-conjugated avidin added as a secondstage reagent. Final detection uses a substrate that undergoes a colorchange in the presence of the peroxidase. Alternatively, the secondaryantibody conjugated to a fluorescent compound, e.g. fluorescein,rhodamine, Texas red, etc. The absence or presence of antibody bindingmay be determined by various methods, including spectrophotometricdetection, fluorimetry, radiography, scintillation counting, etc.

[0072] Quantification can be carried out using any known method,including, but not limited to, enzyme-linked immunosorbent assay(ELISA); radioimmunoassay (RIA); and the like. In general, quantitationis accomplished by comparing the level of ADMA detected in the samplewith the amount of ADMA present in a standard curve.

[0073] In many embodiments, an assay will employ a specific antibody(e.g., an antibody specific for ADMA, or an antibody specific fordimethylarginines), which antibody is bound to a solid support, such asa test strip. Test strips are in provided in a variety of shapes (e.g.,rectangles, squares, circles, etc.) and materials (e.g. nylon, polyvinylpyrollidone, polyester, polycarbonate, cellulose acetate,polyethersulfone, nitrocellulose, and the like). Such assays can bedesigned in any of a number of ways. In general, a specific antibody isbound to a test strip, and the antibody bound to the test strip capturesADMA in the sample. For example, a sample is applied to one end of atest strip, and the components of the sample are allowed to migrate bycapillary action or lateral flow. Methods and devices for lateral flowseparation, detection, and quantitation are known in the art. See, e.g.,U.S. Pat. Nos. 5,569,608; 6,297,020; and 6,403,383. Once the ADMA iscaptured by the bound antibody, a second antibody that is detectablylabeled is used to detect the captured ADMA.

[0074] In another embodiment, a sample that has been modified asdescribed above is spotted onto a membrane (e.g., nylon, polyvinylpyrollidone, polyester, polycarbonate, cellulose acetate,polyethersulfone, nitrocellulose, and the like). Typically, severalspots that correspond to increasing dilutions of the sample are applied.For example, serial 1:2 dilutions are made and spotted onto themembrane. Detectably labeled antibody specific for ADMA or detectablylabeled antibody specific for dimethylarginines is used to quantitatethe level of ADMA in the sample.

[0075] In other embodiments, an assay will employ an antibody specificfor modified SDMA, which antibody is bound to a solid support. Forexample, a test strip is used that includes, in order from a first endto a second end, a sample loading region, a first antibody region thatincludes an antibody specific for modified SDMA, and a second regionthat includes an antibody that binds ADMA (either an antibody specificfor ADMA or an antibody specific for dimethylarginines). A sample isapplied to the sample loading region, and the components of the sampleare allowed to migrate toward the second end of the test strip bycapillary action or lateral flow. Modified SDMA is captured in the firstregion, leaving ADMA free to migrate to the second region, where it iscaptured. The captured ADMA is detected as described above.

[0076] In other embodiments, an assay will employ an antibody specificfor modified SDMA and an antibody specific for modified arginine, whichantibodies are bound to a solid support. For example, a test strip isused that includes, in order from a first end to a second end, a sampleloading region, a first antibody region that includes an antibodyspecific for modified SDMA, a second region that includes an antibodythat binds modified arginine, and a third region that includes anantibody that binds ADMA (either an antibody specific for ADMA or anantibody specific for dimethylarginines). A sample is applied to thesample loading region, and the components of the sample are allowed tomigrate toward the second end of the test strip by capillary action orlateral flow. Modified SDMA is captured in the first region, modifiedarginine is captured in the second region, leaving ADMA free to migrateto the third region, where it is captured. The captured ADMA is detectedas described above.

[0077] As an alternative, the antibody specific for modified SDMA andmodified arginine can be combined into a first region. For example, atest strip is used that includes, in order from a first end to a secondend, a sample loading region, a first antibody region that includes anantibody specific for modified SDMA and an antibody that binds modifiedarginine; and a second region that includes an antibody that binds ADMA(either an antibody specific for ADMA or an antibody specific fordimethylarginines). A sample is applied to the sample loading region,and the components of the sample are allowed to migrate toward thesecond end of the test strip by capillary action or lateral flow.Modified SDMA and modified arginine are captured in the first region,leaving ADMA free to migrate to the second region, where it is captured.The captured ADMA is detected as described above.

[0078] Other Assays

[0079] Other methods of detecting ADMA include liquidchromatography-tandem mass spectrometry. See, e.g., Vishwanathan et al.(2000) J. Chromatogr. B. Biomed. Sci. Appl. 748:157-166.

[0080] Assays for detecting ADMA can also be carried out on a samplethat is depleted of modified SDMA and modified arginine. In theseembodiments, antibodies specific for modified SDMA and antibodiesspecific for modified arginine are used to remove modified SDMA andmodified arginine from a sample that has been reacted with anα-dicarbonyl compound, as described above. Once modified SDMA andmodified arginine are removed from the sample, leaving ADMA, the ADMA isdetected using any known method, including the above-mentioned methods.For example, antibodies specific for modified SDMA and antibodiesspecific for modified arginine are coupled to an insoluble support(e.g., immobilized), and, after reacting the biological sample with theα-dicarbonyl compound, the modified sample is contacted with theimmobilized antibodies. After a suitable time, the modified sample isseparated from the immobilized antibodies, and ADMA is detected in thesample. Antibodies can be immobilized on any of a variety of insolublesupports, including, but not limited to, beads, including magneticbeads, polystyrene beads; an affinity column matrix; a membrane; aplastic surface; and the like.

Antibodies

[0081] The present invention further provides antibodies thatspecifically bind ADMA. Antibodies that specifically bind ADMA do notdetectably bind arginine, SDMA, modified arginine, or modified SDMA, orbind only at a background level. Antibodies specific for ADMA are usefulfor detecting ADMA in a sample that may comprise ADMA, SDMA, andarginine.

[0082] Alternatively, antibodies specific for modified SDMA and modifiedarginine are used together with antibodies directed againstdimethylarginines (after modification of SDMA in the sample, onlyunmodified ADMA would be detected by these antibodies). Accordingly, theα-dicarbonyl modification can be used to enhance the specificity of anyantibody binding to ADMA.

[0083] The present invention further provides antibodies thatspecifically bind modified SDMA, where the guanidino nitrogen residuesare modified by reaction with an α-dicarbonyl compound, as describedabove. Antibodies that specifically bind modified SDMA do not detectablybind arginine, ADMA, or unmodified SDMA, or bind only at a backgroundlevel.

[0084] The present invention further provides antibodies thatspecifically bind modified arginine, where the guanidino nitrogenresidues are modified by reaction with an α-dicarbonyl compound, asdescribed above.

[0085] In many embodiments, a subject antibody is isolated, e.g., is inan environment other than its naturally-occurring environment. Suitableantibodies are obtained by immunizing a host animal with ADMA, withmodified SDMA, or with modified arginine, as appropriate. Suitable hostanimals include mouse, rat sheep, goat, hamster, rabbit, etc.

[0086] For preparation of polyclonal antibodies, the first step isimmunization of the host animal with the antigen, i.e., ADMA, modifiedSDMA, or modified arginine, where the antigen will preferably be insubstantially pure form, comprising less than about 1% contaminant. Toincrease the immune response of the host animal, the antigen istypically coupled to a carrier, and may be combined with an adjuvant,where suitable adjuvants include alum, dextran, sulfate, large polymericanions, oil & water emulsions, e.g. Freund's adjuvant, Freund's completeadjuvant, and the like. The antigen is typically conjugated to a carriermolecule, e.g., a synthetic carrier molecule protein, a syntheticantigen, keyhole limpet hemocyanin, and the like. A variety of hosts maybe immunized to produce the polyclonal antibodies. Such hosts includerabbits, guinea pigs, rodents, e.g. mice, rats, sheep, goats, and thelike. The antigen is administered to the host, e.g., intradermally, orintraperitoneally, with an initial dosage followed by one or more,usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

[0087] Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. The antibody may be purified from the hybridoma cellsupernatants or ascites fluid by conventional techniques, e.g. affinitychromatography using protein bound to an insoluble support, protein Asepharose, etc.

[0088] The antibody may be produced as a single chain, instead of thenormal multimeric structure. Single chain antibodies are described inJost et al. (1994) J.B.C. 269:26267-73, and others. DNA sequencesencoding the variable region of the heavy chain and the variable regionof the light chain are ligated to a spacer encoding at least about 4amino acids of small neutral amino acids, including glycine and/orserine. The protein encoded by this fusion allows assembly of afunctional variable region that retains the specificity and affinity ofthe original antibody.

[0089] Also provided are “artificial” antibodies, e.g., antibodies andantibody fragments produced and selected in vitro. In some embodiments,such antibodies are displayed on the surface of a bacteriophage or otherviral particle. In many embodiments, such artificial antibodies arepresent as fusion proteins with a viral or bacteriophage structuralprotein, including, but not limited to, M13 gene III protein. Methods ofproducing such artificial antibodies are well known in the art. See,e.g., U.S. Pat. Nos. 5,516,637; 5,223,409; 5,658,727; 5,667,988;5,498,538; 5,403,484; 5,571,698; and 5,625,033.

[0090] Also of interest in certain embodiments are humanized antibodies.Methods of humanizing antibodies are known in the art. The humanizedantibody may be the product of an animal having transgenic humanimmunoglobulin constant region genes (see for example InternationalPatent Applications WO 90/10077 and WO 90/04036). Alternatively, theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (see WO 92/02190).

[0091] The use of Ig cDNA for construction of chimeric immunoglobulingenes is known in the art (Liu et al. (1987) Proc. Natl. Acad. Sci. USA.84:3439 and (1987) J. Immunol. 139:3521). mRNA is isolated from ahybridoma or other cell producing the antibody and used to produce cDNA.The cDNA of interest may be amplified by the polymerase chain reactionusing specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions genes may be found in Kabat et al. (1991) Sequences ofProteins of Immunological Interest, N.I.H. publication no. 91-3242.Human C region genes are readily available from known clones. The choiceof isotype will be guided by the desired effector functions, such ascomplement fixation, or activity in antibody-dependent cellularcytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of thehuman light chain constant regions, kappa or lambda, may be used. Thechimeric, humanized antibody is then expressed by conventional methods.Other methods for preparing chimeric antibodies are described in, e.g.,U.S. Pat. No. 5,565,332.

[0092] Antibody fragments, such as Fv, F(ab′)₂ and Fab may be preparedby cleavage of the intact protein, e.g. by protease or chemicalcleavage. Alternatively, a truncated gene is designed. For example, achimeric gene encoding a portion of the F(ab′)₂ fragment would includeDNA sequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

[0093] Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

[0094] Expression vectors include plasmids, retroviruses, YACs, EBVderived episomes, and the like. A convenient vector is one that encodesa functionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

[0095] In some embodiments, a subject antibody is detectably labeled. Adetectable label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Detectable labels include radioisotopes; enzymes whoseproducts are detectable (e.g., luciferase, β-galactosidase, and thelike); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine,phycoerythrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu,or others of the lanthanide series, attached to the antibody throughmetal chelating groups such as EDTA; chemiluminescent compounds, e.g.,luminol, isoluminol, acridinium salts, and the like; bioluminescentcompounds, e.g., luciferin, aequorin (a green fluorescent protein); amagnetic bead; colloidal gold or colored glass or plastic beads (e.g.,polystyrene, polypropylene, latex, etc.) or other labels that can bedetected by mass spectroscopy, NMR spectroscopy, or other analyticalmeans known in the art.

[0096] A subject antibody can be labeled with a fluorescent protein asdescribed in Matz et al., Nature Biotechnology (October 1999)17:969-973; a green fluorescent protein (GFP), including a “humanized”GFP; a GFP from Aequoria victoria or fluorescent mutant thereof, e.g.,as described in U.S. Pat. No. 6,066,476; 6,020,192; 5,985,577;5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445; 5,874,304, thedisclosures of which are herein incorporated by reference; a GFP fromanother species such as Renilla reniformis, Renilla mulleri, orPtilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle etal. (2001) J. Protein Chem. 20:507-519; “humanized” recombinant GFP(hrGFP) (Stratagene); other fluorescent dyes, e.g., coumarin and itsderivatives, e.g. 7-amino-4-methylcoumarin, aminocoumarin, bodipy dyes,such as Bodipy FL, cascade blue, fluorescein and its derivatives, e.g.fluorescein isothiocyanate, Oregon green, rhodamine dyes, e.g. texasred, tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g.Cy3 and Cy5, macrocyclic chelates of lanthanide ions, e.g. quantum dye,etc., chemilumescent dyes, e.g., luciferases.

[0097] In some embodiments, a subject antibody is labeled with anindirectly detectable label. An indirectly detectable label includes amember of a specific binding pair. Specific binding pairs include, butare not limited to, biotin-avidin, biotin-streptavidin, digoxin andantidigoxin and the like.

[0098] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, e.g., in various dipstick assays, conjugated gold oftenappears pink, while various conjugated beads appear the color of thebead.

[0099] In some embodiments, a subject antibody is coupled (directly orthrough a linker) to an insoluble support. The antibody may be attached(coupled) to an insoluble support, including, but not limited to, aplastic surface (e.g., a polystyrene plate); a membrane (e.g.,nitrocellulose, nylon, polyvinyl pyrollidone, polyester, polycarbonate,cellulose acetate, polyethersulfone, etc); a bead (e.g., a magneticbead, a plastic bead); a colloidal particle; an affinity column matrix;and the like.

[0100] Detection using a subject antibody involves use of direct labelsor indirect labels. Indirect labels include second antibodies specificfor a subject antibody (e.g., specific for a heavy chain constant regionof a subject antibody), wherein the second antibody is labeled asdescribed above; and members of specific binding pairs, e.g.,biotin-avidin, and the like. The biological sample may be brought intocontact with an immobilized on a solid support or carrier, such as amembrane, beads, and the like, that is capable of immobilizing cells,cell particles, or soluble proteins. The support may then be washed withsuitable buffers, followed by contacting with a detectably-labeledsubject antibody. Detection methods are known in the art and will bechosen as appropriate to the signal emitted by the detectable label.Detection is generally accomplished in comparison to suitable controls,and to appropriate standards.

Kits

[0101] The present invention further provides kits for practicing thesubject methods. A subject kit will include an α-dicarbonyl compound formodifying the guanidino nitrogen groups of SDMA and arginine; and anantibody. Suitable antibodies include those that bind specifically toADMA; antibodies that specifically bind dimethylarginines (e.g.,antibodies that bind both SDMA and ADMA); antibodies that specificallybind modified SDMA; antibodies that bind α-dicarbonyl forms of SDMA andα-dicarbonyl forms of L-arginine; and antibodies that specifically bindmodified arginine. The subject kit components are typically present in asuitable storage medium, e.g., buffered solution, typically in asuitable container. A subject kit may further include membranes forcarrying out an immunological assay, e.g., a test strip.

[0102] In addition to the above components, the subject kits willfurther include instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, compact disk (CD), digital versatile disk, etc., onwhich the information has been recorded. Yet another means that may bepresent is a website address which may be used via the internet toaccess the information at a removed site. Any convenient means may bepresent in the kits.

Utility

[0103] The subject methods and kits are useful for detecting ADMA in abiological sample. The subject methods are useful for determining alevel of ADMA in a biological sample, and therefore are useful indiagnostic methods for various disorders, including methods fordetermining the risk of developing a disorder.

[0104] The subject methods are useful for diagnosing various disordersfor which elevated ADMA levels are diagnostic, including, but notlimited to, hypertension, hyperhomocysteinemia, hyperglycemia,hypercholesterolemia, insulin resistance, renal insufficiency,congestive heart failure, atherosclerosis, transplant arteriopathy, andendothelial dysfunction and the like. For example, an increase in thelevel of ADMA, compared to the level in a normal, healthy individual,indicates that the individual is at risk for vascular dysfunction ordisease.

[0105] The subject methods are also useful for determining the extent,the severity, the progression, or stage, of a disorder for which anelevated ADMA level is diagnostic. A biological sample is taken at asingle time point and the level of ADMA is compared to a chart ofstandard normal values for ADMA. The severity of the disorder isassessed by comparing the detected levels of ADMA in the biologicalsample with levels of ADMA in a standard curve, and associating thelevel with the severity of the disorder. The severity of the disordermay allow the selection of more efficacious therapies, for example amild elevation of ADMA in a hypercholesterolemic subject may indicatethat lifestyle changes are sufficient therapy, whereas a severeelevation of ADMA would indicate that drug therapy should be employed.

[0106] The subject methods are useful for monitoring progression of adisorder for which an elevated ADMA level is diagnostic. DeterminingADMA levels at different times is used to monitor the progression of thedisorder. A biological sample is taken from the individual and tested ata frequency of once per week, twice weekly, once per month, bimonthly,once every three months, once every four months, once every 6 months, oronce a year, depending on various factors. In these embodiments, thelevel of ADMA in a test sample is compared to the level of ADMA in aprevious sample(s). An increase in the level of ADMA in a test sample,compared to one or more previous test samples, indicates that thedisease is increasing in severity. The rate of increase in the level ofADMA is an indication of the rate of progression of the disease. Areduction in ADMA may be seen with treatment (e.g., insulin-resistantsubjects having elevated ADMA levels exhibit reduced ADMA levelsfollowing treatment with metformin).

[0107] The subject methods are also useful for determining the risk thatan individual will develop a disorder for which an elevated ADMA levelis diagnostic. An elevated ADMA level, compared to a control value for anormal healthy individual, may indicate that the individual is at riskfor developing a disorder for which elevated ADMA levels are diagnostic.ADMA has been shown to be predictive of cardiovascular mortality inpatients with coronary artery disease or renal insufficiency (Zoccali C,Bode-Boger S, Mallamaci F, Benedetto F, Tripepi G, Malatino L,Cataliotti A, Bellanuova I, Fermo I, Frolich J, Boger R. Plasmaconcentration of asymmetrical dimethylarginine and mortality in patientswith end-stage renal disease: a prospective study. Lancet. 2001 Dec.22-29;358(9299):2113-7. Valkonen V P, Paiva H, Salonen J T, Lakka T A,Lehtimaki T, Laakso J, Laaksonen R. Risk of acute coronary events andserum concentration of asymmetrical dimethylarginine. Lancet. 2001 Dec.22-29;358(9299):2127-8.) Additional tests may be recommended todetermine whether an individual is developing a given disorder. In viewof the test results, an appropriate treatment regimen may berecommended.

[0108] The subject methods are also useful for determining the responseof an individual to treatment for a disorder for which an elevated ADMAlevel is diagnostic. Measurements of ADMA levels are used to determinewhether a patient is responding to treatment. ADMA levels are measuredbefore and after a treatment to determine if the treatment isefficacious. ADMA levels are also determined during the course of thetreatment, to determine whether the treatment slows the progression ofthe disease, and to what extent the treatment slows the progression ofthe disease.

EXAMPLES

[0109] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric.

Example 1 Method for Modifying SDMA and Arginine

[0110] There are two isoforms of dimethylarginine—asymmetric (SDMA) andsymmetric (ADMA), depending on how the methyl groups are distributed onthe guanidino function group of arginine. Any detection method for ADMAneeds to be able to distinguish among ADMA, SDMA, and arginine, whichare structurally very similar. Some HPLC system can achieve theresolution; however, it suffers from the drawbacks mentioned above.Available antibodies against dimethylarginine, though improvingefficiency and sensitivity, indiscriminately bind to both ADMA and SDMA(e.g., ab413, Abcam, Cambridge Science Park, UK). The following approachutilizes a chemical reaction that specifically modifies SDMA, but notADMA. SDMA, but not ADMA, will react with α-dicarbonyl compounds,leaving the modified SDMA to sufficiently differ from ADMA such that anantibody directed against dimethylarginines can then be used toselectively detect ADMA. This method also enhances the selectivity ofantibodies or engineered molecules that preferentially (but notselectively) detect ADMA.

[0111] A large number of α-dicarbonyl compounds (dialdehydes,ketoaldehydes and diketones) have been used in the past four decades tomodify arginine residue in proteins. Before site-specific mutationbecame available, such modification indicates whether an arginineresidue is part of an enzyme's active site. Phenylglyoxal (FIG. 1) is anα-dicarbonyl compound still in use today for active site determination.Under mild conditions, phenylglyoxal reacts with the arginine residue.Acting as nucleophiles, the guanidino nitrogens from arginine attack thecarbonyl carbons, forming a five-member ring structure. The unstabledialcohol intermediate then reacts with another phenylglyoxal, givingrise to a product that contains two phenylglyoxal moieties per guanidinogroup (FIG. 2).

[0112] This product is relatively stable at acidic pH's, but at pH>10,the reaction was observed to be reversible. Typically, phenylglyoxal isdissolved in water and the pH is adjusted to 9.0 with 1M NaOH. Asolution containing phenylglyoxal is then added to the sample in such away that the stock solution is diluted 10-fold. The reaction proceeds inthe dark at room temperature for 60-180 minutes (Reference: Tawfik D S,Walter J M, Modification of arginine side chains withp-hydroxyphenylglyoxal, The Proteins Protocol Handbook 2002, 2^(nd)edition, Humana Press Inc.)

[0113] In addition to reacting with the guanidino amine of arginine,phenylglyoxal has been reported to react with the α-amino group of thepeptides to give α-keto acyl peptides. In the context of free aminoacid, this observation indicates that phenylglyoxal will react with allα-amino group of all amino acid. To ensure that phenylglyoxal onlyreacts with the guanidino nitrogen, we can protect the α-amino groupwith a conventional labeling dye such as fuoro-nitro-benzoxadiazole(NBD-F). Many labeling dye molecules are conjugated to the amino acid orproteins by binding to the α-amino group.

[0114] After the α-amino group of SDMA and ADMA are protected byreacting with NBD-F, phenylglyoxal would be added to the mixture. Sincethe guanidino nitrogens on SDMA each take up one methyl group, they bothstill possess a hydrogen free to participate in nucleophilic reaction.ADMA, on the other hand, has both methyl groups occupying the sameguanidino nitrogen, disabling that nitrogen from further reaction (FIG.3).

[0115] Participation of both guanidino nitrogens in the reaction withphenylglyoxal is crucial for the ring formation, and hence stability, ofthe final product. Thus, SDMA, but not ADMA, would react withphenylglyoxal, via the reaction outlined in FIG. 4.

[0116] According to the above scheme, phenylglyoxal would react withboth SDMA and arginine. An antibody against dimethylarginines could beused to specifically detect ADMA, by first reacting the samples withphenylglyoxal, which modifies SDMA, but not ADMA.

[0117] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of detecting asymmetric dimethylarginine(ADMA) in a sample comprising ADMA, symmetric dimethylarginine (SDMA),and arginine, the method comprising the steps of: a) contacting a samplewith an α-dicarbonyl compound, wherein said sample is suspected ofcontaining ADMA and at least one of SDMA and arginine, said contactingresulting in modification of the guanidino nitrogens of SDMA and theguanidino nitrogens of arginine, to produce modified SDMA and modifiedarginine; b) detecting ADMA in the sample.
 2. The method of claim 1,wherein said α-dicarbonyl compound is phenylglyoxal.
 3. The method ofclaim 1, further comprising the step of modifying the α-amino group ofSDMA, ADMA, and arginine before the step of modifying the guanidinonitrogens of SDMA and the guanidino nitrogens of arginine.
 4. The methodof claim 3, wherein the α-amino group is modified with a dye thatprovides a detectable signal.
 5. The method of claim 1, wherein saiddetecting step comprises contacting the sample with an antibody thatbinds specifically to dimethylarginines, wherein said antibody does notbind to the modified SDMA.
 6. The method of claim 1, wherein saiddetecting step comprises contacting the sample with an antibody thatbinds specifically to ADMA.
 7. The method of claim 5, wherein theantibody is detectably labeled.
 8. The method of claim 1, wherein saidADMA is detected by high performance liquid chromatography.
 9. Themethod of claim 1, wherein said ADMA is detected by capillaryelectrophoresis.
 10. An antibody that binds specifically to asymmetricdimethylarginine.
 11. The antibody of claim 10, wherein said antibody isdetectably labeled.
 12. An antibody that binds specifically to modifiedsymmetric dimethylarginine (SDMA), wherein the guanidino nitrogens ofthe SDMA are modified by reaction with an α-dicarbonyl compound.
 13. Akit for detecting asymmetric dimethylarginine (ADMA) in a sample, thekit comprising: an α-dicarbonyl agent that modifies the guanidinonitrogens of SDMA and the guanidino nitrogens of arginine; and anantibody that binds to ADMA.
 14. The kit of claim 13, further comprisingan antibody that binds α-dicarbonyl-modified SDMA, and an antibody thatbinds α-dicarbonyl-modified L-arginine.